565 DOI http://dx.do.org/10.5851/kosfa.2013.33.5.565
Synthesis of Galactooligosaccharides in the Cheese Whey-based Medium by a Lactase from Lactobacillus paracasei YSM0308
Tae-Suk Song, Kyung-Sang Lee, Seung-Bum Kang, Seong-Ho Yoo, Jong-Ik Lee1, Sung-Sik Yoon*
Division of Biological Science and Technology, Yonsei University, Wonju 220-710, Korea
1Samik Dairy and Food Co, Ltd., Seoul 135-841, Korea
Abstract
An enzyme β-galactosidase or β-galactohydrolase [EC3.2.1.23], commonly called lactase, mediates galacto-oligosaccha- ride (GOS) synthesis under conditions of high substrate concentrations. Also, lactase hydrolyzes β(1→4) lactose into glucose and galactose, the latter is successively transferred to free lactose to make various oligosaccharides via transgalactosylation.
GOS is non-digestible to human digestive enzymes and has been used as a functional prebiotics. Among the 24 lactic acid bacteria (LAB) strains used, Lactobacillus paracasei YSM0308 was selected based on its exhibition of the highest β-galac- toside hydrolysis activity, and the crude lactase was prepared for examination of reaction conditions to affect the GOS syn- thesis. Lactase activity was measured with a spectrophotometer using ONPG (o-nitropheyl β-D-galactopyranoside) method.
Lactase activity was not detected in the culture supernatant and was mostly present in the cell pellet after centrifugation.
Activity of the crude lactase preparation ranges from102 to 1,053 units/mL, with the highest activity determined for L. para- casei YSM0308. Optimal conditions for GOS synthesis are as follows: concentration of whey powder, pH, temperature, and time were 30%, pH 6.5-7.0, 30oC, and 4 h, respectively. The final GOS concentration was 19.41% (w/v) by the crude YSM0308 lactase, which was obtained from strain YSM0308 grown in the 10% (w/v) reconstituted whey-based medium.
Key words: cheese whey-based medium, galacto-oligosaccharides (GOS), lactase, Lactobacillus paracasei, transgalacto- sylation activity
Introduction
Whey is opaque greenish yellow with total solids gen- erally of 6.0 to 6.5%, commonly obtained as a by-product from the natural cheese making process, and amount of its production tends to increase as the cheese consump- tion is on the rise, allegedly due to westernization of eat- ing habits in Korea. Fluid whey is rich nutritionally, retaining 52% of the nutrients of the whole milk used Cheddar cheese making. Yield of whey production is approximately 90% (Kosikowski, 1997). Liquid whey contains various useful substances including proteins, lac- tose, vitamins, minerals, essential amino acids, lactic acid, and various enzymes. It exerts several useful func- tionalities such as emulsification, foam formation, gas formation, gelatinization, and solubility (Park et al., 1998).
Despite the favorable nutrient balance of whey, it has
been long neglected in industrial use.
As far as relative sweetness is concerned, oligosaccha- rides are low, compared to sucrose or fructose, which are commonly used sweetener in food industry. Therefore, oligosaccharides are frequently applied to manufacture the low-sweet foods by taking advantage of their low sweetness. Of the oligosaccharides commercially available, well known are malto-oligosaccharides (MOS), isomalto- oligosaccharides (ISO), galacto-oligosaccharides (GOS), fructo-oligosacchrides (FOS), and soybean oligosacccha- rides. They are utilized as a carbon source by few tooth- decaying germs, and able to inhibit the growth of harmful bacteria in the animal intestine by lowering the gut pH. In addition, oliosaccharides are recommended for hyper-gly- cemic patients, and used in the manufacture of infant for- mula, soft drinks, ice cream, bread, pharmaceuticals, and animal feeds as well (Boehm and Stahl, 2007).
GOS are the galactose-containing oligosaccharides of the form Glu α1–[β Gal 1–]n, where n = 2, meaning that 2 units of galactose are attached to glucose. They are usu- ally produced from the concentrated lactose syrup using transgalactosylase activity of the enzyme β-galactosidase.
*Corresponding author: Sung-Sik Yoon, Division of Biological Science and Technology, Yonsei University, Wonju 220-710, Korea. Tel: 82-33-760-2251, Fax: 82-33-760-5576, E-mail:
[email protected] ARTICLE
Earlier studies on oligosaccharides have described about its desirable functions related to nutrient syntheses, pro- motion of metabolism, boost of immuno-modulation, and improved constipation (Kang et al., 1996; Kim and chae, 1997). Despite the most oligosaccharides are distributed as a natural constituent in the plants, its efficient produc- tion process was established using a bacterial lactase from carbo- hydrates, representing sucrose, lactose, glu- cose, and starch. An enzyme responsible to GOS produc- tion is β-galactosidase, simply called lactase. This lactase hydrolyzes β(1→4) glycosides into monosaccharide form of glucose and gal- actose. Resulting galactose from lac- tose hydrolysis is in turn transferred to free lactose to make various oligosacchrides via transgalactosylation (Park and Oh, 2010). GOS is non-digestible to human digestive enzymes, discovered relatively abundant in human milk. Currently, it is mostly used as a prebiotic ingredient in the fermented food in- dustry. β-Galactosi- dase is widely distributed in microorganism including bacteria, yeast, and mold. Among bacterial sources, Bacillus circulans (Fujimoto et al., 1998), B. megaerium (Li et al., 2009), Bifidobacterium adolescentis (Hinz et al., 2004), Bif. infantis (Hung and Lee, 2002), Entero- bacter agglomerans (Lu et al., 2007), L. acidophilus (Nguyen et al., 2006), L. reuteri (Nguyen et al., 2007), and Streptococcus pneumonia (Jeong et al., 2009) have been reported earlier. Yeast lactase was also studied from Cryptococcus laurentii (Ohtsuka et al., 1990), Kluy- ver- omyces fragilis (Ladero et al., 2002), K. lacits (Kim et al., 2003), and Sterigmatomyces elviae (Onishi and Tanaka, 1998). As for mould lactase, Aspergillus aculeatus (van Casteren et al., 2000), Asp. Oryzae (Todorova-Balvay et al., 2006), Bullera singularis (Cho et al., 2003), Sporobo- lomyces singularis (Ishikawa et al., 2005) were published.
This experiment was conducted to examine the condi- tions for GOS synthesis in the reconstituted cheese whey- based medium, which is edible for human consumption, by a lactase from Lactobacillus paracasei YSM0308.
Materials and Methods
Chemicals and media
Dried whey (sweet whey) obtained from Cheddar cheese process was supplied from Samik Dairy Ltd. (Korea). For making growth medium for LAB, the whey powder was completely reconstituted at 10% (w/v) or proper concen- trations in distilled water and partially digested by pro- tease Alcalase (Novo Nordisk, Denmark) in the shaking water bath (Vision Scientific, Korea). The resulting hydro-
lysate was supplemented in common with 5% yeast ex- tract, 0.1% sodium citrate, 1% Tween 80. Final pH was adjusted with 1 N-NaOH to 6.0 and then autoclaved at 90oC for 20 min. MRS medium (BD, USA) and other chemicals were purchased from Sigma (USA), unless specified otherwise.
Quantitative analysis of medium constituents General compositions in whey and reconstituted cheese whey-based medium was determined with Milkoscan II FT120 (Foss, Denmark).
LAB strains
Primary screening experiment was carried out to isolate the LAB strains utilizing a lactose as a sole carbon source.
Total 24 strains include L. paracasei YSM0308, L. helve- ticus FBT1, and 27 strains of LAB collections in this lab, (L. acidophilus KCCM32820, Lactococcus (Lc.) lactis ATCC11454, L. rhamnosus KCCM32405, L. brevis KC- CM11904, L. confusus KCCM40015, L. reuteri ATCC 23272, L. delbrueckii KCCM35468, L. plantarum KCCM 12116, Leuconostoc mesenteriodes KCCM11324, L.
rhamnosus KCCM32405 L. bulgaricus KCCM35463, Weissella kimchii KCCM 41287, and Bif. infantis KCTC 3249), and selected 12 strains based on their lactase activ- ity. Each of 12 strains was cultured for inoculation before transferred in the MRS broth two times.
Growth and viable cell counts
Bacterial cell count was done on both 10% (w/v) recon- stituted whey-based medium and the MRS agar medium, if necessary, enumerated the colonies grown on the sur- face of the medium at 48 h incubation. Growth curve was drawn by based on viable cell counts in the samples taken at different time points up to 60 h of incubation at 37oC.
Assay for the crude lactase activity
Test strains were transferred for activation in 10 mL of MRS broth and grown at 30oC overnight and then inocu- lated into 100 mL of 10% (w/v) reconstituted whey me- dium at same temperature for 18 h. Cultured broth was centrifuged at 2,000 g for 20 min. The cell pellet was com- pletely suspended in the original volume for washing.
After draining off the supernatant, the pellet was com- pletely dissolved in 20 mL distilled water, which was mixed with a lysozyme stock solution (1 mg/mL) in order to induce cell lysis, followed by incubation at 37oC for about 60 min. Crude lactase solution was prepared by son- icating (VCX130, Sonics, USA) the lysozyme-treated cul-
ture 30 times at every 10 s on ice-bath. Lactase activity was assayed by a slight modification previously described by Nguyen et al. (2006), determined amount of o-nitro- phenol liberated from ONPG (Sigma) per min. Composi- tions of reaction mixture are as follows; 1.4 mL of 100 mM phosphate buffer (pH 7.0), 500µL crude lactase solu- tion, 100µL ONPG stock solution (20 mM). After incu- bation at 30oC for 10 min, 200µL Na2CO3 (0.2 M) was added to stop the reaction, yellow color was measured at 405 nm using a spectrophotometer (Shimadzu, model UV- 1601PC). Lactase unit is defined as amount of µmole o- nitrophenol (ONP) liberated from ONPG per min. Enzyme unit was calculated from the standard curve of o-nitro- phenol (Fig. 1).
GOS assay on thin-layer chromagtography (TLC)
TLC method by Itoh et al. (1982) was slightly modified in order to determine GOS production by lactase under defined conditions. 15 mL of 30% (w/v) whey medium prepared as described above was mixed with 5 mL of crude lactase solution (206 U/mg protein), and incubated at 30oC for 4 h. Samples were properly diluted and spotted on TLC (Merck, Germany) for GOS production. Develop- ing solution was a mixture of isopropanol and water (4:1) and color was developed by treating the solution mixed equal amount (5 mL) of aniline (dissolved in 1% acetone) and 5 mL diphenylamine (1% dissolved in acetone) with 1 mL of 85% phosphoric acid.
Analysis of the GOS
For HPLC determination, 15 mL sample was mixed with active carbon powder (Active Carbon, Ilsan) for brief boil- ing, and then the cooled samples were filtered with a fil- ter paper (Whatman No.2). Samples filtered again by sy- ringe filter (0.45µm, Advantec, Japan) was subjected to inject to the High Performance Liquid Chromatography equipment (HPLC, e2695, Waters, USA) with RI detector and YMC-Pack Polyamine II column.
Results and Discussion
Screening of lactase activity in LAB strains
For total 12 LAB strains grown in MRS broth, lactase activity was measured by ONPG disc method for both cell pellet and culture supernatant (Table 1). Lactase acti- Fig. 1. Standard curve of o-nitrophenol in 100 mM Na-phos-
phate buffer (pH 7.0).
Table 1. Detection of lactase activity of LAB strains grown on MRS agar
List of LAB stocks Activity
Culture Supernatant Cell Pellet
A Lactobacillus rhamnosus KCCM32405 − +
B Lactobacillus brevis KCCM11904 − +
C Lactobacillus confusus KCCM40015 − +
D Lactobacillus reuteri ATCC23272 − +
E Lactobacillus delbrueckii KCCM35468 − +
F Lactobacillus acidophilus KCCM32820 − +
G Lactobacillus plantarum KCCM12116 − +
H Lactococcus lactis ATCC11454 − +
I Leuconostoc mesenteriodes KCCM11324 − −
J Weissella kimchii KCCM41287 − −
K Lactobacillus helveticus FBT1 − +
L Lactobacillus paracasei YSM0308 − +
+, positive; −, negative
Table 2. Comparisons of lactase activity of the commercial LAB starters
LAB strains L. acidophilus Lc. lactis L. helveticus L. paracasei
Enzyme activity (U)1) 228 102 520 1,053
1)Enzyme activity is defined as the transformation of 1 mol of the o-nitrophenol per minute under standard conditions.
vity was only found in the cell pellet but no lactase activ- ity in the culture supernatant, indicating this lactase is intracelluar which is synthesized inside the cell without excretion out of the cells. Lactase activity was found in all LAB strains except two strains of Leuconostoc mesen- teriodes KCCM11324 and Lactobacillus GG, both were confirmed lactose-negative (data not shown). Nguyen et al. (2007) disrupted the bacterial cell wall with French Press (Amico, USA) for extracting a lactase from L. aci- dophilus.
Selection of LAB strain for GOS synthesis
The 14% (w/v) cheese whey powder medium contains 1.70% protein, 9.84% lactose, and 1.02% ash. In order to induce lactase, all LAB strains were cultivated in the 14%
whey medium (pH 6.0), in which yeast extract is indis- pensable for growth of LAB as suggested by Rech et al.
(1999). By using ONPG method, lactase activity was determined for the primary isolates along with four LAB strains currently used as commercial yogurt starter strains As shown in Table 2, their lactase activities ranged from 102 units to max 1,053 units, the latter was highest which was produced by L. paracasei YSM0308.
Simple assay of GOS synthesis on TLC
As shown in Fig. 2, TLC chromatogram has shown that few spots responding to GOS were clearly positioned on the sample lanes (5-8) just below the monosaccharide spots of glucose, galactose, and lactose as a control (lane 7 to 8). These smeared spots explains because the GOS in general consists inherently of 3-mer to 5-mers (Lee et al., 2011). Protein concentration of the L. paracasei cell-free lysate was measured at 5.1 mg/mL in the crude lactase solution, with specific activity of 206 U/mg protein. Ac- cording to Nguyen et al. (2006, 2007), protein concentra-
tion and specific activity of L. acidophilus and L. reuteri were 230 mg/mL and 180 units/mg, respectively.
Effect of pH on L. paracasei lactase activity As shown in Fig. 3, YSM0308 lactase activity based on the ONPG method was optimal at pH 6.5-7.0. Previous works have described that optimal pH for Bif. infantis lactase was 6.5 (Hung et al., 2002), pH 6.0 for Bif. ado- lescentis lactase (Hinz et al., 2004), pH 6.5-8.0 for L. aci- dophilus lactase (Nguyen et al., 2007).
Effect of lactose concentrations on GOS synthesis Initial lactose concentration is by far the most signifi- cant factor affecting GOS formation (Boon et al., 2000) as the GOS was synthesized using lactose by an enzyme lactase. Fig. 4 has shown that GOS increased with the whey concentration up to 40%, followed by being slow down in the production rate at the higher than 30% of whey concentrations. GOS production increased with inc- reasing lactose conversion until a maximum was reached at approximately 50% conversion rate. Before the lactose conversion reached 40%, there was a constant increase in GOS concentration as well as increased in glucose and galactose concentrations. However, at higher conversion rates, there was a shift in the reactions to favor the hydro- lysis, which resulted in increased formation of monosac- charides (glucose and galactose) and decreased amounts of GOS. Carlos et al. (2012) reported that synthesis of GOS with Aspergillus oryzae galactosidase were maxi- Fig. 2. Detection of GOS synthesis by a thin layer chromatog-
raphy (TLC) method. Lane 1: glucose, Lane 2: galac- tose, Lane 3: lactose.
Fig. 3. Effect of pH on L. paracasei YSM0308 lactase activity.
*Enzyme activity is defined as the transformation of 1µmol of the o-nitrophenol per minute under stan- dard conditions.
mally obtained at 50% (w/w) lactose monohydrate of ini- tial concentration at 47.5oC.
Effect of temperature on GOS synthesis
Incubation temperature is known as one of critical fac- tors affecting the enzyme-mediated transgalactosylation reactions. For GOS synthesis by a YSM0308 lactase, tem- perature was adjusted from 20 to 60oC by 10oC increment, and the amount of GOS was determined in the medium at the predetermined temperatures. As the results shown in Fig. 5, maximum synthesis was obtained at 30oC and gradually decrease at the temperature above. This result indicated that optimal temperature of GOS synthesis is a little lower than that of growth for strain YSM0308 in the 30% cheese whey medium. According to a previous work,
optimal temperature for GOS synthesis with L. reuteri lac- tase was 30oC (Splechtna et al., 2006). They also reported that the temperatures higher than 37oC was not possible due to the lack of enzyme thermostability. As for transga- lactosylation temperature using a lactase from Geobacil- lus stearothermophilus KVE39 (Placier et al., 2009), the reaction were optimal at 37oC in 0.1 M potassium phos- phate buffer, pH 6.5. The result was very similar to the transgalactosylation of L. paracasei YSM0308 lactase in this study.
Effect of reaction time on GOS synthesis
Incubation time affect significantly the yields of GOS synthesis, which was determined during incubation up to 8 h at 30oC. L. paracasei YSM0308 lactase has steadily produced galacto-oligosaccharides when its production was monitored at every two hours up to 8 h, confirmed a steady increase for the first 4 h and then slightly increased Fig. 4. Effect of powdered whey concentrations on GOS pro-
duction by L. paracasei YSM0308 lactase at 30oC for 4 h.
Fig. 5. Effect of temperature on GOS synthesis by L. paracasei YSM0308 lactase in 30% whey-based medium for 4 h.
Fig. 6. Effect of reaction time on GOS synthesis by L. para- casei YSM0308 lactase in 30% whey-based medium at 30oC.
Fig. 7. HPLC chromatogram of GOS synthesis.
(Fig. 6). Therefore, 8 hr incubation is thought to be optimal in terms of cost-effective production.
HPLC Determination of GOS by L. paracasei lactase For determination of galacto-oligosaccharides, HPLC analysis was performed (Fig. 7). As average amount of lac- tose in whey is about 30%, GOS is synthesized through a mechanism that free lactose molecule binds to free galac- tose molecule via transgalactosylation reaction. In this experiment using 30% whey medium, lactose level dec- reased, glucose and galactose increase, and peaks corre- sponding to GOS were confirmed on the chromatogram during the passage of time. As shown in Table 3, maxi- mum production was obtained from 19.41% (w/v). The total GOS yield from 500 g/L of lactose under the opti- mal conditions was about 32%, which corresponded to 160.4 g/L of GOS (Lee et al., 2011). Lee et al. (2011) reported that GOS synthesized using active E. coli β- galactosidase was mostly galactosyl lactose, indicating that a galactose moiety was most likely transferred to a lactose molecule during GOS synthesis.
To conclude, GOS production from lactose by L. para- casei YSM0308 β-galactosidase was mainly affected by the lactose concentrations in the reconstituted whey me- dium. More GOS could be produced in the presence of higher lactose concentrations. Temperature and pH signif- icantly affected the overall reaction rate. Therefore, fur- ther study needs to increase GOS synthesis in the recon- stituted whey-based medium.
Acknowledgement
This work was fully supported by a grant (No. 2012-7- 5023) of the high value-added food products project in Ministry of Agriculture, Fisheries, and Foods, 2010.
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Total GOS(%)
1 0.76 5.13 1.66 80.73 1.70 3.59 1.24 4.65 12.84
2 0.47 2.41 1.42 86.7 0 4.13 0.47 4.01 10.03
3 0.51 6.21 1.74 81.58 0.23 4.10 1.37 3.94 11.38
4 0.56 6.60 2.07 80.07 0 3.95 1.84 4.61 12.47
5 0 5.38 1.56 82.33 1.80 3.65 1.10 4.18 12.29
6 0 4.06 5.04 79.81 2.20 4.05 1.14 3.70 16.13
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12 2.12 8.95 0.73 76.95 1.8 3.39 0.74 5.14 11.8
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(Received 2013.1.15/Revised 2013.7.19/Accepted 2013.8.22)
